Polyketides represent a large family of diverse compounds synthesized from 2-carbon units through a series of condensations and subsequent modifications. Polyketides occur in many types of organisms, including fungi and mycelial bacteria, in particular, the actinomycetes. There are a wide variety of polyketide structures, and the class of polyketides encompasses numerous compounds with diverse activities. Erythromycin, FK-506, FK-520, megalomicin, narbomycin, oleandomycin, picromycin, rapamycin, spinocyn, and tylosin are examples of such compounds. Given the difficulty in producing polyketide compounds by traditional chemical methodology, and the typically low production of polyketides in wild-type cells, there has been considerable interest in finding improved or alternate means to produce polyketide compounds. See PCT publication Nos. WO 93/13663; WO 95/08548; WO 96/40968; 97/02358; and 98/27203; U.S. Pat. Nos. 4,874,748; 5,063,155; 5,098,837; 5,149,639; 5,672,491; 5,712,146; and 5,962,290; and Fu et al., 1994, Biochemistry 33: 9321-9326; McDaniel et al., 1993, Science 262: 1546-1550; and Rohr, 1995, Angew. Chem. Int. Ed. Engl. 34(8): 881-888, each of which is incorporated herein by reference.
Polyketides are synthesized in nature by polyketide synthase (PKS) enzymes. These enzymes, which are complexes of multiple large proteins, are similar to the synthases that catalyze condensation of 2-carbon units in the biosynthesis of fatty acids. PKS enzymes are encoded by PKS genes that usually consist of three or more open reading frames (ORFs). Two major types of PKS enzymes are known; these differ in their composition and mode of synthesis. These two major types of PKS enzymes are commonly referred to as Type I or “modular” and Type II “iterative” PKS enzymes. A third type of PKS found primarily in fungal cells has features of both the Type I and Type II enzymes and is referred to as a “fungal” PKS enzymes.
Modular PKSs are responsible for producing a large number of 12-, 14-, and 16-membered macrolide antibiotics including erythromycin, megalomicin, methymycin, narbomycin, oleandomycin, picromycin, and tylosin. Each ORF of a modular PKS can comprise one, two, or more “modules” of ketosynthase activity, each module of which consists of at least two (if a loading module) and more typically three (for the simplest extender module) or more enzymatic activities or “domains.” These large multifunctional enzymes (>300,000 kDa) catalyze the biosynthesis of polyketide macrolactones through multistep pathways involving decarboxylative condensations between acyl thioesters followed by cycles of varying β-carbon processing activities (see O'Hagan, D. The polyketide metabolites; E. Horwood: New York, 1991, incorporated herein by reference).
During the past half decade, the study of modular PKS function and specificity has been greatly facilitated by the plasmid-based Streptomyces coelicolor expression system developed with the 6-deoxyerythronolide B (6-dEB) synthase (DEBS) genes (see Kao et al., 1994, Science, 265: 509-512, McDaniel et al., 1993, Science 262: 1546-1557, and U.S. Pat. Nos. 5,672,491 and 5,712,146, each of which is incorporated herein by reference). The advantages to this plasmid-based genetic system for DEBS are that it overcomes the tedious and limited techniques for manipulating the natural DEBS host organism, Saccharopolyspora erythraea, allows more facile construction of recombinant PKSs, and reduces the complexity of PKS analysis by providing a “clean” host background. This system also expedited construction of the first combinatorial modular polyketide library in Streptomyces (see PCT publication Nos. WO 98/49315 and 00/024907, each of which is incorporated herein by reference).
The ability to control aspects of polyketide biosynthesis, such as monomer selection and degree of β-carbon processing, by genetic manipulation of PKSs has stimulated great interest in the combinatorial engineering of novel antibiotics (see Hutchinson, 1998, Curr. Opin. Microbiol. 1: 319-329; Carreras and Santi, 1998, Curr. Opin. Biotech. 9: 403-411; and U.S. Pat. Nos. 5,712,146 and 5,672,491, each of which is incorporated herein by reference). This interest has resulted in the cloning, analysis, and manipulation by recombinant DNA technology of genes that encode PKS enzymes. The resulting technology allows one to manipulate a known PKS gene cluster either to produce the polyketide synthesized by that PKS at higher levels than occur in nature or in hosts that otherwise do not produce the polyketide. The technology also allows one to produce molecules that are structurally related to, but distinct from, the polyketides produced from known PKS gene clusters.
There has been a great deal of interest in expressing polyketides produced by Type I and Type II PKS enzymes in host cells that do not normally express such enzymes. For example, the production of the fungal polyketide 6-methylsalicylic acid (6-MSA) in heterologous E. coli, yeast, and plant cells has been reported. See Kealey et al., January 1998, Production of a polyketide natural product in nonpolyketide-producing prokaryotic and eukaryotic host, Proc. Natl. Acad. Sci. USA 95:505-9, U.S. Pat. No. 6,033,883, and PCT Patent Publication Nos. 98/27203 and 99/02669, each of which is incorporated herein by reference. Heterologous production of 6-MSA required or was considerably increased by co-expression of a heterologous acyl carrier protein synthase (ACPS) and that, for E. coli, media supplements were helpful in increasing the level of the malonyl CoA substrate utilized in 6-MSA biosynthesis. See also, PCT Patent Publication No. 97/13845, incorporated herein by reference.
The biosynthesis of other polyketides requires substrates other than or in addition to malonyl CoA. Such substrates include, for example, propionyl CoA, 2-methylmalonyl CoA, 2-hydroxymalonyl CoA, and 2-ethylmalonyl CoA. Of the myriad host cells possible for utilization as polyketide producing hosts, many do not naturally produce such substrates. Given the potential for making valuable and useful polyketides in large quantities in heterologous host cells, there is a need for host cells capable of making the substrates required for polyketide biosynthesis. The present invention helps meet that need by providing recombinant host cells, expression vectors, and methods for making polyketides in diverse host cells.